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• W3092838799 abstract "Article14 October 2020Open Access Source Data BAZ2A safeguards genome architecture of ground-state pluripotent stem cells Damian Dalcher Damian Dalcher Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Jennifer Yihong Tan Jennifer Yihong Tan orcid.org/0000-0003-1064-1162 Department of Computational Biology, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Cristiana Bersaglieri Cristiana Bersaglieri Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Rodrigo Peña-Hernández Rodrigo Peña-Hernández Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Eva Vollenweider Eva Vollenweider Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Stefan Zeyen Stefan Zeyen Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Marc W Schmid Marc W Schmid Service and Support for Science IT, University of Zurich, Zurich, Switzerland Search for more papers by this author Valerio Bianchi Valerio Bianchi Oncode Institute, Hubrecht Institute-KNAW, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Stefan Butz Stefan Butz Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Marcin Roganowicz Marcin Roganowicz Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Rostyslav Kuzyakiv Rostyslav Kuzyakiv Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Service and Support for Science IT, University of Zurich, Zurich, Switzerland Search for more papers by this author Tuncay Baubec Tuncay Baubec orcid.org/0000-0001-8474-6587 Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Search for more papers by this author Ana Claudia Marques Ana Claudia Marques orcid.org/0000-0001-5174-8092 Department of Computational Biology, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Raffaella Santoro Corresponding Author Raffaella Santoro [email protected] orcid.org/0000-0001-9894-2896 Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Search for more papers by this author Damian Dalcher Damian Dalcher Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Jennifer Yihong Tan Jennifer Yihong Tan orcid.org/0000-0003-1064-1162 Department of Computational Biology, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Cristiana Bersaglieri Cristiana Bersaglieri Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Rodrigo Peña-Hernández Rodrigo Peña-Hernández Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Eva Vollenweider Eva Vollenweider Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Stefan Zeyen Stefan Zeyen Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Marc W Schmid Marc W Schmid Service and Support for Science IT, University of Zurich, Zurich, Switzerland Search for more papers by this author Valerio Bianchi Valerio Bianchi Oncode Institute, Hubrecht Institute-KNAW, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Stefan Butz Stefan Butz Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Marcin Roganowicz Marcin Roganowicz Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland Search for more papers by this author Rostyslav Kuzyakiv Rostyslav Kuzyakiv Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Service and Support for Science IT, University of Zurich, Zurich, Switzerland Search for more papers by this author Tuncay Baubec Tuncay Baubec orcid.org/0000-0001-8474-6587 Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Search for more papers by this author Ana Claudia Marques Ana Claudia Marques orcid.org/0000-0001-5174-8092 Department of Computational Biology, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Raffaella Santoro Corresponding Author Raffaella Santoro [email protected] orcid.org/0000-0001-9894-2896 Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland Search for more papers by this author Author Information Damian Dalcher1,2, Jennifer Yihong Tan3, Cristiana Bersaglieri1,2, Rodrigo Peña-Hernández1,2, Eva Vollenweider1,2, Stefan Zeyen1,2, Marc W Schmid4, Valerio Bianchi5, Stefan Butz1,2, Marcin Roganowicz1,2, Rostyslav Kuzyakiv1,4, Tuncay Baubec1, Ana Claudia Marques3 and Raffaella Santoro *,1 1Department of Molecular Mechanisms of Disease, DMMD, University of Zurich, Zurich, Switzerland 2Molecular Life Science Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland 3Department of Computational Biology, University of Lausanne, Lausanne, Switzerland 4Service and Support for Science IT, University of Zurich, Zurich, Switzerland 5Oncode Institute, Hubrecht Institute-KNAW, University Medical Center Utrecht, Utrecht, The Netherlands *Corresponding author. Tel: +41 44 63 55475; E-mail: [email protected] The EMBO Journal (2020)39:e105606https://doi.org/10.15252/embj.2020105606 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground-state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub-domains within the active A compartment, which intersect through long-range contacts. We found that ground-state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A-dependent manner. Finally, ground-state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity. Synopsis How genome organization is regulated in embryonic stem cells (ESC) according to cell state and chromatin structure remains elusive. Here, comparison of basic, ground-state to developmentally advanced mouse ESCs reveals nucleolar remodeling complex (NoRC) component BAZ2A/TIP5 as a critical regulator of genome partitioning and cell identity. BAZ2A binds at active and open genome and supports growth of ground-state but not advanced ESCs. BAZ2A interacts with SNF2H, TOP2A and cohesin on ESC chromatin. BAZ2A associates with chromatin sub-domains within the active A-compartment that intersectthrough long-range contacts. BAZ2A deletion perturbs gene expression and histone H3K27 trimethylation in ground-state ESCs. BAZ2A limits invasion of ESC active chromatin into inactive domains. Introduction The 3D genome organization is functionally important for correct execution of gene expression programs. Chromosomes have an intrinsic tendency to segregate into compartments based on the local epigenetic landscape and transcriptional activity. At the megabase scale, the genome can be divided into two compartments, called A and B compartments, which are estimated by an eigenvector analysis of the genome contact matrix after normalization by the observed-expected method (Lieberman-Aiden et al, 2009). Genomic contacts predominately occur between loci belonging to the same compartment. Compartment A is highly enriched for open and active chromatin whereas compartment B is enriched for closed and repressed chromatin. Furthermore, long-distance contacts have been shown to occur between domains with similar chromatin states (Rao et al, 2014). Removal or depletion of chromatin-associated cohesin eliminates all loop domains and increased compartmentalization (Haarhuis et al, 2017; Nora et al, 2017; Rao et al, 2017; Schwarzer et al, 2017). The opposite effect was achieved by increasing the residence time of cohesins on DNA, which leads to extension of chromatin loops and a less strict segregation between A and B compartments with a decrease of far-cis contacts (Haarhuis et al, 2017). In contrast to topologically associating domains (TAD) boundaries, which are generally conserved among cell types, A and B compartments are cell type specific (Rao et al, 2014; Dixon et al, 2015; Bonev et al, 2017). However, the mechanisms underlying these changes and their impact on gene expression and epigenetic states remain yet unclear. Pluripotent embryonic stem cells (ESCs) provide an exceptional system to address this question as they exist in a variety of states that represent distinct chromatin and epigenetic features. In vitro, ESC types are largely defined by culture conditions, which mimic the natural development of the embryo from the blastocyst to post-implantation stages (Hackett & Surani, 2014). ESCs can be propagated in medium containing fetal calf serum and leukemia inhibitory factor (LIF; ESC+serum) or in serum-free 2i medium (ESC+2i) that contains LIF plus two small-molecule kinase inhibitors for MEK/ERK (PD0325901) and GSK3 (CHIR99021; Ying et al, 2008). Both ESC+2i and ESC+serum are pluripotent. ESC+2i closely resemble the developmental ground-state in vivo whereas ESC+serum are developmentally advanced (Boroviak et al, 2014). These two pluripotent cell types display distinct chromatin and epigenetic features. At the nucleosomal level, the chromatin configuration is more open in ESC+2i than in ESC+serum (Ricci et al, 2015). Furthermore, the epigenetic and chromatin organization of ESC+2i is in a less repressed state. ESC+2i have hypomethylated DNA similar to pre-implantation embryos, whereas ESC+serum genome is hypermethylated, reminiscent of post-implantation embryos (Marks et al, 2012; Ficz et al, 2013; Habibi et al, 2013; Leitch et al, 2013). Similarly, there is a reduced prevalence of the repressive H3K27me3 mark at Polycomb target promoters in ESC+2i (Marks et al, 2012; Joshi et al, 2015). Recent studies showed that ESC+serum contain a set of extremely long-range interactions that depend on Polycomb repressive complexes 1 and 2 (PRC1, PRC2; Joshi et al, 2015; Schoenfelder et al, 2015). In contrast, ESC+2i do not contain these long-range PRC interactions and instead exhibit chromatin decompaction at PRC target loci (Joshi et al, 2015; McLaughlin et al, 2019). In this work, we made use of these two closely and developmentally related ESC types to elucidate how genome organization and compartmentalization are regulated according to cell state and chromatin structure. We show that only the active and open genome of ground-state ESCs rely on BAZ2A (also known as TIP5), a factor that together with SNF2H (SMARCA5) constitutes the nucleolar remodeling complex NoRC (Santoro et al, 2002). BAZ2A interacts on ESC chromatin with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. We found that BAZ2A associates with sub-domains within the active A compartment that strongly intersect through long-range contacts in ESCs. BAZ2A specifically regulates the highly open chromatin of ESC+2i by limiting the invasion of active domains into repressive compartments. Depletion of BAZ2A specifically affects ground-state ESCs by increasing chromatin accessibility at chromatin regions including the repressed B compartments, which in turn decrease their repressive features such as upregulation of gene expression and acquisition of active epigenetic marks. Furthermore, the binding of BAZ2A with active chromatin domains of ground-state ESCs regulates H3K27me3 genome occupancy, a process that also involves TOP2A. Finally, the ground-state-specific role of BAZ2A was also evident by impaired differentiation capacity and deregulation of genes linked to developmental process, which all occur only upon BAZ2A depletion in ESC+2i whereas ESC+serum remained unaffected. Our results suggest that chromatin remodeling and topoisomerase activities might serve to counteract the intrinsic propensity of the highly open and active chromatin of ground-state ESCs to invade repressive domains. This effort in controlling open/active chromatin domains is required to establish active and repressed genome partitioning and preserves cell function and identity. Results Ground-state and developmentally advanced pluripotent ESCs differ in the requirement of BAZ2A for cell proliferation and differentiation capacity The nucleolar remodeling complex NoRC consists of two subunits, the ATPase SNF2H and BAZ2A, a >200 kDa protein that shares sequence homology with the largest subunits of SNF2H/ISWI-containing remodeling complexes (Strohner et al, 2001; Santoro et al, 2002). In differentiated cells, BAZ2A is mainly localized in nucleoli, associates with ribosomal RNA (rRNA) genes and establishes their epigenetic silencing (Santoro et al, 2002; Mayer et al, 2006; Guetg et al, 2010). In contrast, in ESCs, recruitment of BAZ2A to rRNA genes, its ability to silence rRNA genes, and its nucleolar localization are impaired through a long non-coding RNA-mediated mechanism (Savic et al, 2014; Leone et al, 2017). Our previous work and initial analysis suggested that in ESCs BAZ2A function was not related to rRNA transcriptional control. First, BAZ2A is more highly expressed in ESC+2i than in differentiated cells (Fig 1A). Second, although not bound to rRNA genes, BAZ2A is still tightly associated with chromatin in ESC+2i (Fig 1B). Third, and consistent with previous results (Savic et al, 2014), we observed a decrease in cell number of ESC+2i upon BAZ2A depletion by siRNA (Fig 1C and D). Similar results were obtained with a different siRNA and in another ESC line (Appendix Fig S1A and B). BAZ2A downregulation in ESC+2i induced a moderate arrest at G1 phase of cell cycle without any evident sign of apoptotic cell death (Appendix Fig S1C–E). Fourth, and in line with previous results (Savic et al, 2014), after induction of monolayer differentiation upon withdrawal of LIF, ESC+2i treated with siRNA-Baz2a underwent cell death while control cells displayed morphological structures typical of differentiated cells and were negative for alkaline phosphatase staining (Fig 1F and G). All these results indicated that ground-state ESCs depend on BAZ2A expression for proliferation and differentiation capacity and highlighted an unexpected non-nucleolar function of BAZ2A. Next, we asked whether developmentally advanced ESC+serum were also dependent on BAZ2A. Surprisingly, although BAZ2A expression levels and knockdown efficiency in ESC+2i and ESC+serum were similar (Fig 1C and E), depletion of BAZ2A in ESC+serum did not cause any evident defect in proliferation or differentiation (Fig 1D,F, and G), suggesting ground-state-specific role of BAZ2A. To further support these results, we performed CRISPR/Cas9 to generate Baz2a-KO directly in ESC+serum and ESC+2i. While we were able to generate Baz2a-KO lines in ESC+serum (Fig 1H), all our attempts to establish Baz2a-KO directly in ESC+2i failed, a result that is consistent with the proliferative defects observed upon siRNA-mediated BAZ2A depletion only in ESC+2i, but not in ESC+serum (Fig 1H). Cell morphology, proliferation, and differentiation capacity were similar between control and Baz2a-KO ESC+serum (Fig 1I and J, Appendix Fig S1F). However, during the transition from serum to 2i conditions Baz2a-KO cells lose self-renewal and proliferation capacity with substantial cell death (Fig 1J, Appendix Fig S1G), indicating that BAZ2A is specifically essential in ground-state ESCs. Figure 1. BAZ2A is required for proliferation and differentiation of ESC+2i BAZ2A expression is higher in ESC+2i than in differentiated cells (neural progenitors, NPC). Left panel. BAZ2A mRNA levels were measured by qRT–PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d., and statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Right panel. Western blot showing BAZ2A protein levels in ESC+2i and NPC. Tubulin is shown as a protein loading control. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by Western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control. siRNA-knockdown efficiency of BAZ2A shown by qRT–PCR and Western blot. BAZ2A mRNA levels were measured by qRT–PCR and normalized to Rps12 mRNA and to each ESC line. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (**** < 0.0001). Right panel. Western blot showing BAZ2A protein levels. Tubulin is shown as a protein loading control. BAZ2A knockdown affects proliferation of ESC+2i but not of ESC+serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01; ns, non-significant). BAZ2A is expressed at similar levels in both ESC+2i and ESC+serum. Left panel. BAZ2A mRNA levels were measured by qRT–PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. Right panel. Western blot showing BAZ2A protein levels in ESC+2i and ESC+serum. Tubulin and histone H3 are shown as a protein loading controls. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF. Quantification of differentiated cells. Values represent relative number of differentiated cells from three independent experiments relative to control cells. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001; ns, non-significant). Western blot showing BAZ2A protein levels in two BAZ2A-KO ESC lines obtained via CRISPr/Cas9 directly in ESC+serum. PARP1 is shown as a protein loading control. Cell proliferation is not affected in BAZ2A-KO ESC+serum. Data represent relative cell numbers 2 days culture starting with the same number of cells. Average values of four independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns, non-significant). Representative images of wt and Baz2a-KO ESC+serum before and after transition in 2i conditions. Source Data for Figure 1 [embj2020105606-sup-0010-SDataFig1.pdf] Download figure Download PowerPoint Taken together, these results highlighted a substantial difference in the requirement of BAZ2A for cell proliferation and differentiation capacities between ground-state and developmentally advanced pluripotent ESCs. BAZ2A regulates gene expression in ground-state ESCs To elucidate the ground-state-specific role of BAZ2A, we analyzed gene expression of ESC+2i and ESC+serum treated with siRNA-Baz2a or siRNA-control (Fig 2A, Table EV1). We found that depletion of BAZ2A induces greater differential gene expression in ESC+2i than in ESC+serum. Upon BAZ2A depletion in ESC+2i, 1934 genes showed transcriptional changes (log2 fold change > 0.58; P<0.05; BAZ2A 2i-regulated genes: 1236 upregulated and 698 downregulated). In contrast, ESC+serum depleted for BAZ2A showed only moderate changes in gene expression compared with ESC+2i, and the total number of genes affected by BAZ2A knockdown was almost four times less (351 upregulated and 207 downregulated; Fig 2A). Only a minority of differentially expressed genes were upregulated (65) or downregulated (56) by BAZ2A knockdown in both ESC states (Fig EV1A). Validation by qRT–PCR, using two siRNAs and a different ESC line, supported a role of BAZ2A in the regulation of gene expression in ESC+2i but not in ESC+serum (Figs 2B and EV1B and C). Gene ontology analysis revealed that differentially expressed genes in ESC+2i are enriched in pathways linked to developmental processes (Fig 2C, Table EV2). In contrast, genes differentially expressed in ESC+serum were mostly implicated in biological processes linked to cell signaling and cell adhesion. Figure 2. BAZ2A regulates gene expression specifically in ESC+2i Volcano plot showing fold change (log2 values) in transcript level of ESC+2i and ESC+serum with two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Gene expression values of three replicates were averaged and selected for 1.5 fold changes and P < 0.05. Statistical significance (P-values) was calculated using R package DEseq2. Validation by qRT–PCR of genes regulated by BAZ2A in ESC+2i but not in ESC+serum (upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red). Nanog, Rex1, and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001). Data without P-values are statistically non significant. Top 10 biological process gene ontology terms as determined using DAVID for genes regulated by BAZ2A in ESC+2i and ESC+serum. Source Data for Figure 2 [embj2020105606-sup-0011-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. BAZ2A regulates gene expression specifically in ESC+2i Venn diagrams showing number of differently expressed genes upon BAZ2A knockdown detected in ESC+2i compared with ESC+serum. qRT–PCR. Expression analysis of BAZ2A regulated genes in ESC+2i upon BAZ2A knockdown using a different siRNA-Baz2a (siRNA-Baz2a#2). Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, and downregulated genes are in red. mRNA levels were normalized to Rps12 mRNA and to ESC+2i transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non-significant, *<0.05, ***<0.001, ****<0.0001). qRT–PCR. Expression analysis of BAZ2A-regulated genes in another ESC line (ESC#2) cultured in 2i medium upon BAZ2A knockdown. mRNA levels were normalized to Rps12 mRNA and to ESC+2i transfected with siRNA-Control. Experiment performed as one replicate. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, and downregulated genes are in red. BAZ2A knockdown does not affect proliferation of TKO-ESCs cultured in serum/LIF. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non-significant). Knockdown of BAZ2A in TKO-ESCs cultured in serum (ESC-TKO +serum) does not affect transcription of genes regulated by BAZ2A in ESC+2i. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, and downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, **** < 0.0001). Data without P-values are statistically non significant. BAZ2A knockdown does not affect proliferation of Eed−/− and Ring1b−/− ESCs cultured in serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test. (ns: non-significant). BAZ2A knockdown does affect expression of genes regulated by BAZ2A in Eed−/− and Ring1b−/− ESCs cultured in serum. qRT–PCR experiments in Eed−/− (left panel) and Ring1b−/− (right panel) -ESCs cultured in serum treated with siRNA-control or siRNA-Baz2a. mRNA levels were normalized to Rps12 mRNA and ESC+2i transfected with siRNA-Control. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, and downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (ns: non-significant, ***< 0.001). Download figure Download PowerPoint Next, we asked whether the role of BAZ2A in the regulation of gene expression specifically in ESC+2i was a consequence of the chromatin state of these cells (low DNA methylation and low H3K27me3 occupancy at PRC target promoters). To mimic this status in ESC+serum, we analyzed ESCs that lack DNA methyltransferases DNMT1, 3B, and 3A (TKO-ESC) or the core components of PRC1 and PRC2 complexes (Ring1b−/− ESC and Eed−/− ESC). BAZ2A depletion in these ESC-KO lines cultured in serum did not affect cell proliferation or expression of BAZ2A 2i-regulated genes (Fig EV1D–G). These results show that BAZ2A specifically affects gene expression of ESC+2i and suggest that this process might not depend on the low DNA methylation content and low H3K27me3 levels at PRC target promoters characterizing ground-state ESCs. BAZ2A associates with large and active genomic regions of ESCs To determine how BAZ2A specifically regulates gene expression in ground-state ESCs, we measured and compared BAZ2A genomic occupancy between ESC+2i and ESC+serum. We established an ESC line containing a FLAG-HA (F/H) tag at the N-terminus of both Baz2a alleles (Appendix Fig S3A–C). Previous studies showed that the fusion of the F/H peptide at the N-terminus of BAZ2A does not affect its activity (Mayer et al, 2006; Guetg et al, 2012). The obtained ESC lines (F/H-BAZ2A-" @default.
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• W3092838799 title "<scp>BAZ</scp> 2A safeguards genome architecture of ground‐state pluripotent stem cells" @default.
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